Traditional assays to identify Epstein-Barr virus (EBV) associated lymphomas detect viral gene products that may not be expressed when the EBV genome is rearranged or partially deleted. Recent evidence of atypical EBV in lymphomas that were EBV-negative by "gold standard" EBER in situ hybridization assays suggests EBV may contribute to a larger proportion of lymphomas than previously assumed. The goal of the proposed study is to develop and apply a novel set of cost-effective and reliable assays to clarify the presence of EBV in non-Hodgkin's lymphoma (NHL). Specific aims for the R21 phase are to 1) optimize quantitative real-time PCR assays targeting five disparate but highly conserved EBV genes plus the defective W-Zhet rearrangement, 2) use these assays to estimate EBV viral load in archival tissues comparable to samples available for population-based research, 3) use in situ assays to localize infection to neoplastic cells, 4) identify a subset of assays and viral load cut points that reliably identify cases EBV, or remnants of EBV in neoplastic cells, and 5) compare PCR results with EBER-ISH to determine whether traditional methods misclassify EBV-associated cases. Specific aims for the R33 phase are to 1) use the optimized battery of assays to identify neoplastic EBV in archival sections from 163 unselected NHL cases, 2) compare results with EBER-ISH to determine whether the new assays enhance detection of EBV-related tumors, 3) describe relations between somatic mutations and typical and atypical EBV-positive cases and 4) conduct a preliminary analysis of risk factors for typical and atypical EBV-positive NHL. Quantitative PCR assays may be cost-effective for large-scale research, but lack of atypical EBV-positive lymphomas would substantially discount hypotheses regarding hit-and-run pathogenic mechanisms resulting in EBV deletion or rearrangement. Evidence that the novel assays detect new EBV-associated cases would support a large population-based study to confirm results. Improved understanding of EBV and NHL could lead to new approaches to prevent lymphoma, and new methods to diagnose, monitor, and treat lymphoma patients.